Breast cancer is the second leading cause of cancer related deaths in women in the United States, but is the most common cancer among American women.1. Determining the extent of cancer proliferation in patients is still an unmet clinical need. Sometimes, multiple scans and biopsies are required to determine if masses are enlarging or are merely normal for that specific patient. Current scans utilize molecular imaging agents that target over- expressed cell surface receptors on cancerous tissues. These cell surface receptors are also found on non- cancerous tissues in lower abundance. The most commonly used PET radiopharmaceutical, [18F]-FDG, has been used to determine which cells are consuming glucose at a higher rate than others, but also gets taken up in healthy tissues with elevated glucose metabolism. Instead of utilizing cell surface receptors or common metabolic pathways, pH (low) insertion peptide - pHLIP - specifically targets cells with low extracellular pH, which is a common characteristic of all cancer cells.2 PHLIP, a portion of the bacteriorhodopsin protein, has three very different secondary structures at varying pH: 1) an unfolded (non-helical) secondary structure at basic pH; 2) a somewhat more ordered unfolded (non-helical) secondary structure that can interact with cell surfaces at neutral pH; and 3) an alpha-helical secondary structure that can insert into a cell membrane at acidic pH. Labeling of pHLIP with florescent molecules or radioactive metals that can be detected in optical or PET scanners is possible with today's technology. The synthetic pHLIP variations - conjugated to fluorescent molecules or chelators with radioactive metals - must retain the pH dependent secondary structure to allow pHLIP to only target low pH cancer cells. This project is three-fold: 1) improve the design and chemical synthesis of fluorescently labeled pHLIP (pHLIP-FL) agents for use in surgical settings; 2) create new variations that retain the physical properties of the parent pHLIP molecule and include positron emission tomography (PET) imaging agents (pHLIP- PET); and 3) investigate the use of pHLIP in a pretargeting approach (pHLIP-PT) to limit the dose of radioactivity to the kidneys during imaging and therapy. The mentoring and prior successes at Memorial Sloan-Kettering Cancer Center (MSKCC) will provide help in achieving these research goals as well as training to produce a well-rounded and informed independent cancer researcher.